Studies are proposed to test the hypothesis that the induction of Ets transcriptional repressors provides a molecular link between the terminal phase of macrophage differentiation and permanent cell cycle arrest. This hypothesis is based on our recent studies demonstrating that the Ets repressor METS/PE1 is markedly up regulated during terminal macrophage differentiation and can block Ras-dependent cell proliferation without inhibiting Ras-dependent expression of macrophage-specific genes. Selective inhibition of proliferation is proposed to result from the ability of METS to bind as a monomer to Ets sites in a series of E2F-dependent cell cycle-regulatory genes, but not to sites recognized by ternary complexes of Ets activators and AP-1 proteins. METS-dependent inhibition of cell proliferation is hypothesized to require the DEAD-box containing protein DP103, which has also been identified as a target of the EBNA2 and EBNA3c proteins that are required for immortalization of B lymphocytes following Epstein-Barr virus infection. DP103 is proposed to function as a corepressor of METS by nucleating the assembly of a novel co-repressor complex. Biochemical, cellular and molecular genetic approaches will be used to test the hypothesis that functional interactions between the METS/DP103 complex and E2F/pRB family protein complexes are involved in directing permanent exit from the cell cycle during terminal macrophage differentiation. Five Specific Aims are proposed: Specific Aim 1 will test at a genome-wide level the hypothesis that METS selectively replaces Ets activators on cell cycle control genes, but not cell type-specific genes, during terminal macrophage differentiation. Specific Aim 2 will test the hypothesis that METS/DP103-mediated growth inhibition requires interactions with members of the Rb family and associated corepressor complexes that harbor histone deacetylase and histone methyltransferase activities. Specific Aim 3 will test the hypothesis that METS/PE1 and the related Ets repressor ERF negatively regulate macrophage proliferation by analysis of METS and ERF-deficient macrophages. Specific Aim 4 will test the hypothesis that METS and ERF-mediated growth inhibition requires interactions with DP103, through the analysis of DP103- deficient macrophages. Specific Aim 5 will test the hypothesis that cells can become resistant to METS-mediated growth inhibition by exporting METS from the nucleus and/or by inhibiting functional interactions with DP103. These studies are should provide significant new insights into mechanisms by which permanent exit from the cell cycle is achieved during terminal differentiation of macrophages and other cell types.